Application of tomato slsph2 gene in improving heat tolerance of tomato

Abstract

Tomato SlSPRH2 The application of the gene in improving the heat tolerance of tomato contains the amino acid sequence as shown in SEQ ID NO: 2 or an amino acid sequence with at least 95% homology thereof, and the sequence retains the functional properties of regulating the heat tolerance of tomato. The discovery not only enriches our understanding of the heat tolerance mechanism of plants, but also opens up a new gene resource approach for future breeding of new tomato varieties that can resist high temperature stress and maintain high yield and quality, and has important theoretical guiding significance and practical application value.

Description

TECHNICAL FIELD

[0001] The application belongs to the technical field of plant genetic engineering, and relates to application of a tomato gene SlSPRH2 in improving heat resistance of tomatoes. BACKGROUND

[0002] During the growth and development of plants, temperature is one of the key factors affecting whether the plants can grow normally. When plants feel abnormal temperature, changes such as cell dehydration, intracellular osmotic pressure and pH increase, cell structure and plasma membrane system damage, influence of organelle functions such as chloroplasts and mitochondria, and the like occur, and finally abnormal growth and development of the plants is caused (Yamazaki et al., 2009; Yin et al., 2014; Lohani et al., 2020).

[0003] Tomato (Solanum lycopersicum) is a perennial herb of the Solanaceae family, Solanum subgenus Lycopersicon, rich in various vitamins, and has high nutritional and medicinal values. In addition, the tomato genome is small and easy to transform, and the tomato is a commonly used model plant and occupies an important position in fruits and vegetables. The optimal growth temperature of the tomato is about 28°C, and the growth and development of the tomato is extremely susceptible to high temperature. After the tomato is affected by high temperature, many adverse consequences occur, such as affecting normal development of the plant to make the plant prematurely senesce, high temperature burning and damaging the structure and organization of the plant, causing pollen abortion, fertilization failure, and low fruit setting rate to affect yield.

[0004] Plants have acquired a series of mechanisms for responding to external stress during long-term evolution. The mitogen activated protein kinase (MAPK) cascade is an important member among them. The MAPK cascade is a highly conserved important signal module in eukaryotes, and this pathway can amplify and recognize signals of the plant responding to the external environment and transduce them to the inside of the cell (Meng and Zhang 2013), and is an important central node of signal transduction in plant cells. At present, many studies have shown that the MAPK cascade not only plays an important role in the growth and development of plants, but also plays a crucial role in the process of plants adapting to various adverse environments.

[0005] The biological function of MAPK cascade is mainly determined by the downstream target proteins of MAPK phosphorylation. The specific downstream substrates of MAPK include other kinases, enzymes and transcription factors (Joo et al., 2008; Meng et al., 2013; Zhang et al., 2018). MAPK cascade is an important signaling module involved in the whole growth and development process of plants. The diversity of downstream substrates of MAPK cascade enables MAPK cascade to regulate different biological processes. Many studies have shown that MAPK substrates play an important role in the process of plant growth and development, including cytoplasmic division, reproduction and leaf senescence (Soyano, et al. 2003; Wang et al., 2008; Zhou et al., 2009). At the same time, MAPK substrates also play an important role in abiotic stress signal transduction, including plant tolerance to low temperature environment, resistance to high temperature and drought, salt tolerance, etc. (Li et a1., 2017; Evrard et al., 2013; Zhang et al., 2012; Manuka et al., 2018). Therefore, studying the specific downstream substrates of MAPK to improve the heat tolerance of tomato can not only directly promote the development of tomato industry, but also provide experimental ideas and theoretical basis for studying the response mechanism of other plants to high temperature. At the same time, it provides a theoretical basis and practical guidance for enhancing the heat tolerance of tomato and other crops by genetic means. SUMMARY

[0006] The technical problem solved by the present application is to provide the application of tomato SlSPRH2 gene in improving the heat tolerance of tomato. The gene is knocked out by CRISPR / Cas9 method, so as to further realize the application of SlSPRH2 in improving the heat tolerance of tomato.

[0007] Technical scheme: A protein for regulating the heat tolerance of tomato, comprising an amino acid sequence as shown in SEQ ID NO: 2, or an amino acid sequence with at least 95% homology, and the sequence retains the functional characteristics of regulating the heat tolerance of tomato.

[0008] SlSPRH2 gene encoding the above-mentioned protein has a nucleic acid sequence as shown in SEQ ID NO: 1, or a nucleic acid sequence with at least 95% homology, and the gene can encode a protein with the function of regulating the heat tolerance of tomato.

[0009] The application of the above-mentioned protein as a negative regulatory factor in regulating the heat tolerance of tomato.

[0010] The application of the above-mentioned gene as a negative regulatory factor in regulating the heat tolerance of tomato.

[0011] A method for improving the heat tolerance of tomatoes, comprising reducing the expression level of the above-mentioned protein, the method for reducing expression comprising gene silencing, gene mutation, gene knockout or antisense RNA technology.

[0012] A method for breeding heat-tolerant tomato varieties, comprising screening plants with reduced expression of the above-mentioned gene, or introducing overexpression vectors and knockout vector plasmids into tomato plants by genetic transformation.

[0013] A kit for detecting the presence or expression level of the above-mentioned protein or gene in tomatoes, the kit comprising antibodies capable of specifically recognizing the protein or probes capable of specifically binding the gene.

[0014] The above-mentioned protein as a detection marker in the preparation of a biological agent for detecting the regulation of the heat tolerance of tomatoes.

[0015] The above-mentioned gene as a detection marker in the preparation of a transgenic tomato or other biological agent for detecting the regulation of heat tolerance.

[0016] Beneficial effects: The present application selects M14 wild type of tomato variety Ailsa Craig (AC) as the research basis, successfully constructs SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D three kinds of overexpression transgenic tomato materials, and uses CRISPR / Cas9 gene editing technology to accurately obtain mutant plants of SlSPRH2 gene. Through heat tolerance evaluation, including but not limited to plant phenotype observation in high temperature environment, in vitro leaf phenotype analysis, tomato leaf DAB and NBT staining experiment, and detailed change analysis of antioxidant enzyme activity, the present application proves that SlSPRH2 gene plays a key role in significantly improving the heat tolerance of tomatoes. Further, the present application preliminarily reveals the core function and action path of SlSPRH2 gene and its specific Ser-42 phosphorylation site in the complex mechanism of tomatoes coping with high temperature stress, which provides insights for in-depth understanding of the molecular basis of tomato heat tolerance. This discovery not only enriches our understanding of plant heat tolerance mechanism, but also opens up a new gene resource approach for future breeding of new tomato varieties that can resist high temperature stress and maintain high yield and quality, and has important theoretical guidance significance and practical application value. BRIEF DESCRIPTION OF DRAWINGS

[0017] Figure 1 Plasmid map of overexpression vector (A: pBI121-35S:SlSPRH2 plasmid map; B: pBI121-35S:SlSPRH2 S42A plasmid map; C: pBI121-35S:SlSPRH2S42D Plasmid map).

[0018] Figure 2 SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D SlSPRH2-OE positive plant identification (1-6: SlSPRH2-OE positive plant identification; 7-12: SlSPRH2 S42A -OE positive plant identification; 13-15: SlSPRH2 S42D -OE positive plant identification; - is wild type; + is positive plasmid).

[0019] Figure 3 SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D SlSPRH2-OE transgenic tomato relative expression determination (OE-S2109, OE-S2105, OE-S255 is SlSPRH2 overexpression plant phenotype, OE-2A61, OE-2A25, OE-2A35 is SlSPRH2 S42A OE plant phenotype, OE-2D30, OE-2D118, OE-2D101 is SlSPRH2 S42D OE plant phenotype; difference analysis: n=3, * represents p<0.05, ** represents p<0.01, *** represents p<0.001, **** represents p<0.0001).

[0020] Figure 4 SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D SlSPRH2-OE tomato growth phenotype (OE-S2109, OE-S2105, OE-S255 is SlSPRH2 overexpression plant phenotype, OE-2A61, OE-2A25, OE-2A35 is SlSPRH2 W OE plant phenotype, OE-2 W 53, OE-S2 W 55 is SlSPRH2 overexpression plant phenotype, OE-2 A 6, OE-2 A 25, OE-2 A 35 is SlSPRH2 overexpression plant phenotype, OE-2 S42A OE plant phenotype, OE-2 A 102, OE-2 A 118, OE-2 A 101 is SlSPRH2 overexpression plant phenotype). S42D

[0021] Figure 5 ​Wild-type under high temperature stress and SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE detached leaf phenotype (A) and chlorophyll content changes (B) (OE-S2109 and OE-S255 are phenotypes of plants overexpressing SlSPRH2, OE-2A25 and OE-2A35 are phenotypes of plants overexpressing SlSPRH2) S42A Plant phenotype, OE-2D118 and OE-2D101 are overexpression of SlSPRH2 S42D (Plant phenotype).

[0022] Figure 6 Wild-type under high temperature stress and SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE tomato leaf DAB(A) and NBT(B) staining results. (OE-S2109, OE-S255, and OE-S2105 represent the phenotype of plants overexpressing SlSPRH2; OE-2A61, OE-2A25, and OE-2A35 represent the phenotype of plants overexpressing SlSPRH2.) S42A Plant phenotype, OE-2D30, OE-2D118, and OE-2D102 are overexpression of SlSPRH2 S42D (Plant phenotype).

[0023] Figure 7 SlSPRH2-OE and SlSPRH2 under high temperature stress S42A -OE and SlSPRH2 S42D - Changes in the activities of antioxidant enzymes (CAT, APX, GR, POD, and SOD) in leaves of OE and wild-type tomatoes. (OE-S2109, OE-S255, and OE-S2105 represent the phenotype of plants overexpressing SlSPRH2; OE-2A61, OE-2A25, and OE-2A35 represent the phenotype of plants overexpressing SlSPRH2.) S42A Plant phenotype, OE-2D30, OE-2D118, and OE-2D101 are overexpression of SlSPRH2 S42D Plant phenotype. Control: Control group without high-temperature treatment; HT: 45℃, 6h; * represents p<0.05, ** represents p<0.01, *** represents p<0.001).

[0024] Figure 8 Designed for SlSPRH2 gene knockout target.

[0025] Figure 9 Identification of SlSPRH2 homologous and mutant materials.

[0026] Figure 10Growth phenotype of wild type and SlSPRH2 knockout mutant tomato under long-term high temperature stress.

[0027] Figure 11 Leaf phenotype (A) and chlorophyll content change (B) of wild type and SlSPRH2 knockout mutant tomato under high temperature stress.

[0028] Figure 12 NBT (left) and DAB (right) staining results of wild type and SlSPRH2 knockout mutant tomato leaves under high temperature stress.

[0029] Figure 13 Antioxidant enzyme (CAT, APX, POD and SOD) activity change of wild type and SlSPRH2 knockout mutant tomato leaves under high temperature stress (Control: control group without high temperature treatment; HT: 45℃, 6h; * represents p<0.05, ** represents p<0.01, *** represents p<0.001).

[0030] Figure 14 Principle diagram of overlap extension PCR amplification step.

[0031] Figure 15 Vector sequencing results (A) pBI121-SlSPRH2 S42A Vector sequencing results (B) pBI121-SlSPRH2 S42D Vector sequencing results (C). DETAILED DESCRIPTION

[0032] The application will be further described below in conjunction with specific examples.

[0033] Example 1: Overexpression of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Tomato plants are obtained

[0034] The specific operation method is as follows:

[0035] (1) According to the ID of SlSPRH2 gene (Solyc09g089650), the corresponding CDS sequence information was found in the database of Solanaceae (Sol Genomics Network), and the full-length sequence primer was designed by Primer 5.0, and the 5' end of the upstream and downstream primers was added with BamH I and Sac I as the enzyme digestion site and the homologous arm, primer 1: SlSPRH2-pBI121-F: gatgacgatgacaagggatccATGTCAATTAACCACCGGACTTCC (SEQ ID NO. 9); primer 6: SlSPRH2-pBI121-R: atcggggaaatcatagagctcTCACCGTCTATCGGCGGTT (SEQ ID NO. 10).

[0036] Note: The lowercase letters in the primer sequence are the vector homologous arms.

[0037] (2) After designing the primer, the specificity of the primer was determined by comparing in the NCBI database. The cDNA of wild type M14 tomato was used as a template, and the corresponding PCR amplification procedure (Table 2) was used to amplify the target fragment by using a high-fidelity amplification system (Table 1). The complete gene of SlSPRH2 was obtained.

[0038] Table 1 High-fidelity amplification system

[0039]

[0040] Table 2 PCR amplification procedure

[0041]

[0042] (3) SlSPRH2 S42A and SlSPRH2 S42D gene point mutation primer design: taking the CDS sequence of SlSPRH2 gene as the blueprint, SlSPRH2 S42A was obtained by changing TCA (124-126) to GCA (124-126); SlSPRH2 S42D was obtained by changing TCA (124-126) to GAT (124-126). The single-point mutation amplification primers of SlSPRH2 S42A and SlSPRH2 S42D were designed by Novyze single-point mutation primer design website (https: / / crm.vazyme.com / cetool / singlepoint.html mL).

[0043] Primer 3: SlSPRH2 S42A- pBI121-mu-F: 5'-CTCTTTTAgatCCGTTGATTACTTCTCCGGTT T-3' (SEQ ID NO. 13)

[0044] Primer 2: SlSPRH2 S42A - pBI121-mu-R: 5'-CAACGGatcTAAAAGAGGTATAGCCACATTCC AAT-3' (SEQ ID NO. 14)

[0045] Primer 4: SlSPRH2 S42D - pBI121-mu-F: 5'-CTCTTTTAgatCCGTTGATTACTTCTCCGGTT T-3' (SEQ ID NO. 13)

[0046] Primer 5: SlSPRH2 S42D - pBI121-mu-R: 5'-CAACGGatcTAAAAGAGGTATAGCCACATTCC AAT-3' (SEQ ID NO. 14)

[0047] Note: In the primer sequence, the capital letters are the vector homologous arm, and the lowercase letters are the point mutation site.

[0048] (4) After designing the single-point mutation amplification primer, overlap extension PCR was performed as shown in Figure 14 Primer 1 / 2 and primer 3 / 6; primer 1 / 5 and primer 4 / 6 were used for the first round of PCR, and the desired mutation site was introduced into primer 2 / 3 and 4 / 5. After the first round of PCR, the two PCR products were recovered by gel, mixed uniformly, and then primer 1 / 4 was used for the second round of PCR. The product was SlSPRH2 S42A and SlSPRH2 S42D point gene.

[0049] (5) Construction of overexpression vector: pBI121 (as an overexpression vector, directly from the original source: the plasmid was stored in the laboratory) was double digested with BamH I and Sac I restriction enzymes, and the double digestion system is shown in Table 3. After electrophoresis gel recovery, it was ligated with SlSPRH2 and SlSPRH2 S42A and SlSPRH2 S42D gene mentioned above by homologous recombination, and the recombination ligation reaction system is shown in Table 4, 50°C for 30 min. After ligation, the competent cells of E. coli DH5α (Nanjing Qikexing Biological Co., Ltd.) were transformed, plated, and grown overnight. The next day, single colony was picked and bacterial liquid was identified. The positive bacterial liquid with successful identification was sequenced, and the sequencing results are shown in Figure 15The plasmid was extracted after the bacteria were expanded and sequenced.

[0050] Table 3 Double enzyme digestion reaction system

[0051]

[0052] Table 4 Recombination ligation reaction system

[0053]

[0054] (6) Agrobacterium-mediated genetic transformation of tomato: pBI121-SlSPRH2, pBI121-SlSPRH2 S42A pBI121-SlSPRH2 S42D The recombinant plasmid was transformed into Agrobacterium competent cells GV3101 (Nanjing Novozyme BioTech Co., Ltd.), plated, and the bacterial growth was waited for, and single colony was picked up on the third to fourth day, and the bacterial liquid was identified. The positive bacterial liquid identified successfully was used as the Agrobacterium species for subsequent infection of tomato cotyledon. Finally, the genetic transformation of tomato was carried out through related plant tissue culture experiments.

[0055] (7) Positive identification: a specific primer 35S-3'-F: 5'-GACGCACAATCCCACTATCC-3' (SEQ ID NO. 15) was designed. 35S-3'-F and SlSPRH2-pBI121-R were used to identify the tissue culture seedlings by PCR, and the positive plants were screened. Part of SlSPRH2-OE, SlSPRH2 S42A -OE and SlSPRH2 S42D -OE overexpression transgenic tomato positive identification results are shown in Table 4. Figure 2

[0056] Example 2: Overexpression of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D Tomato plant relative expression amount analysis.

[0057] The specific operation method is as follows:

[0058] (1) Fresh overexpression SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D transgenic tomato leaf samples were cut and wrapped with tin foil paper, labeled, rapidly cooled in liquid nitrogen, and stored in a -80°C refrigerator. The total RNA of the sample was extracted (the total RNA of the tomato sample was extracted according to the operation instruction of RNA isolater Total RNA Extraction Reagent (Nanjing Novozyme BioTech Co., Ltd.)).

[0059] ​(2) Reverse transcription synthesis: The first step is to extract RNA from the overexpressed tomato leaves (identified positive plants), and the extracted RNA is mixed in an RNase-free centrifuge tube according to Table 5. After mixing, gently blow the pipette and mix, 42°C, reaction for 2min, the second step is to configure the reverse transcription reaction system: add 4μL of 5×HisScriptⅢQrtSuperMix to the reaction solution of the first step. Perform reverse transcription reaction: 37°C, 15min; 85°C, 5min. The third step is to obtain cDNA.

[0060] Table 5 Genomic DNA removal system

[0061]

[0062] (3) qRT-PCR detection of SlSPRH2-OE, SlSPRH2-OE and SlSPRH2-OE tomato materials according to Table 6 tomato qRT-PCR primers, Table 7 qRT-PCR amplification system and Table 8 qRT-PCR amplification program. S42A S42D The results are shown in Table 6. Figure 3

[0063] Table 6 Tomato qRT-PCR primer design

[0064]

[0065] Table 7 qRT-PCR amplification system

[0066]

[0067] Table 8 qRT-PCR amplification program

[0068]

[0069] (4) The relative expression results show that the expression in the overexpression positive plants is significantly higher than that in the wild type plants. The results show that the overexpression of SlSPRH2-OE, SlSPRH2-OE and SlSPRH2-OE tomato materials is successfully constructed. S42A S42D

[0070] Example 3: Analysis of heat resistance of SlSPRH2, SlSPRH2 and SlSPRH2 overexpressed tomato plants. S42A S42D

[0071] The specific operation method is as follows:

[0072] ​​​​​​(1) Tomato soil phenotype: Wild-type and overexpression tomato plants grown to five-leaf stage, select the same growth of 42℃ / 38℃ (16h / 8h) treatment for one week, then restore to normal conditions and observe the phenotype as shown in Figure 4 The results can be seen that after high temperature treatment, SlSPRH2-OE and SlSPRH2 S42D -OE plants grow worse than wild-type plants, SlSPRH2 S42A -OE plants have no significant difference compared with wild-type plants.

[0073] (2) In vitro leaf phenotype: After high temperature treatment (45℃, 3h) for 5 days, the in vitro leaf was decolorized with acetone, and the chlorophyll content was determined. The results are shown in Figure 5 The chlorophyll content of the in vitro leaf of the overexpression SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants is lower than that of wild-type plants, while the chlorophyll content of SlSPRH2 S42A -OE plants has little difference with wild-type.

[0074] (3) Tomato leaf DAB, NBT staining: Select the same growth of 5-leaf stage wild-type and overexpression positive plants, and perform staining after 45℃ treatment for 6h. The experiment is divided into two groups: control group and high temperature treatment group, each group has 3 biological repeats. The results are shown in Figure 6 NBT staining results show the same as DAB staining results. After high temperature treatment, SlSPRH2-OE and SlSPRH2 S42D -OE tomato plant leaf staining sites are more than wild-type plants, and the staining is deeper, SlSPRH2 S42A -OE has little difference in color compared with wild-type.

[0075] (4) Antioxidant enzyme activity determination: Overexpression positive and wild-type five-leaf stage tomato plants with good growth were sampled (about 3 leaves) at 0h and 45℃ high temperature treatment for 6h, respectively, with 3 biological repeats. Add 1.6mL enzyme liquid extract, grind in liquid nitrogen, centrifuge at 4℃, 12000xg for 30min, and take the supernatant as tomato crude enzyme extract. And determine the activities of APX, CAT, GR, SOD and POD five kinds of antioxidant protective enzymes under the corresponding wavelength, repeat 3 times. The results are shown in Figure 7 The antioxidant enzyme activity of SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants decreases more than wild-type and SlSPRH2 S42A -OE tomato plants.

[0076] (5) After overexpression of SlSPRH2, SlSPRH2S42A and SlSPRH2 S42D Tomato plant heat tolerance analysis found that SlSPRH2-OE and SlSPRH2 S42D -OE tomato plants compared with wild type and SlSPRH2 S42A -OE tomato plants, the former suffered more serious oxidative damage. It showed that SlSPRH2 was negatively regulated in tomato heat tolerance, and Ser-42 was the key phosphorylation site.

[0077] Example 4: SlSPRH2 gene knockout tomato plants were obtained

[0078] The specific operation method is as follows:

[0079] (1) SlSPRH2 sgRNA design and knockout vector construction: SlSPRH2 gene target sequence sgRNA was designed using CRISPR-P2 website (http: / / crispr.hzau.edu.cn / cgi-bin / CRISPR2 / CRISPR): target sequence 1 (sgRNA-1): 5'-GGACTTCCACCGGAGACCGT-3'(SEQ ID NO.3); target sequence 2 (sgRNA-2): 5'- CCACATTCCAATCGGTCGGT-3'(SEQ ID NO.4), SlSPRH2 gene knockout target design as shown in Figure 8 .

[0080] (2) Using kit, first synthesis Oligo, then prepare Oligo dimer: Buffer Anneal 18 μL, UPOligo 1 μL, Low Oligo 1 μL, H2O supplement to 20 μL; after mixing, heat at 95℃ for 3 minutes, then slowly reduce to 20℃ at about 0.2℃ / second; wherein the primer sequence is:

[0081] UP-1 Oligo: gtttGGACTTCCACCGGAGACCGT (SEQ ID NO.5)

[0082] Low-1 Oligo: aaacACGGTCTCCGGTGGAAGTCC (SEQ ID NO.6)

[0083] UP-2 Oligo: gtttCCACATTCCAATCGGTCGGT (SEQ ID NO.7)

[0084] Low-2 Oligo: aaacACCGACCGATTGGAATGTGG (SEQ ID NO.8)

[0085] (3) and the Oligo dimer is connected to the vector BGK015 to obtain a knockout vector: CRISPR / Cas Vector 2 μL, Oligo dimer 1 μL, Enzyme Mix 1 μL, H2O supplemented to 10 μL; mix the components on ice, mix well after mixing, and react at room temperature 20°C for 1 hour. Then the ligation product is transformed into E. coli competent cells DH5α (Nanjing Qikeli Biological Co., Ltd.), plated, grown overnight, and the next day single colony colonies are picked and bacterial liquid is identified. The positive bacterial liquid that is successfully identified is sequenced, and after the bacterial liquid sequencing is confirmed to be correct, the subsequent genetic transformation of tomato is carried out.

[0086] (4) The genetic transformation method of the SlSPRH2 gene knockout tomato plant is consistent with that in Example 1, and the SlSPRH2 gene knockout tomato plant line and mutant material identification results are shown in Table 2. Figure 9

[0087] Example 5: Analysis of heat tolerance of SlSPRH2 gene knockout tomato plants.

[0088] (1) Tomato soil culture phenotype: wild type and SlSPRH2 gene knockout tomato plants growing to five leaf stage, select the same growth 42°C / 38°C (16h / 8h) treatment for 5 days, then recover for 2 days and observe the phenotype as shown in Table 3. Figure 10 The results show that after high temperature treatment, the SlSPRH2 gene knockout tomato plants grow better than the wild type plants.

[0089] (2) In vitro leaf phenotype: after high temperature treatment (45°C, 6h) and recovery for 7 days, the in vitro leaf is decolorized with acetone, and the content of chlorophyll is determined. The results are shown in Table 4. Figure 11 The results show that after high temperature treatment, the SlSPRH2 gene knockout tomato plants grow better than the wild type plants.

[0090] (3) DAB, NBT staining of tomato leaves: select wild type and SlSPRH2 gene knockout tomato plant leaves with the same growth at five leaf stage, and perform staining after 6h treatment at 45°C. The experiment is divided into two groups: control group and high temperature treatment group, with 3 biological replicates in each group. The results are shown in Table 5. Figure 12 The results show that after high temperature treatment, the SlSPRH2 gene knockout tomato plants grow better than the wild type plants.

[0091] ​(4) Anti-oxidase activity determination: well-grown SlSPRH2 gene knockout and wild-type five-leaf stage tomato plants were sampled (about 3 leaves) at 0 h and 45℃ high temperature treatment for 6 h, respectively, with 3 biological replicates. Add 1.6 mL enzyme liquid extraction solution, grind in liquid nitrogen, centrifuge at 4℃, 12000xg for 30 min, and take the supernatant as the tomato crude enzyme solution. The activities of APX, CAT, SOD and POD five kinds of anti-oxidation protective enzymes were determined at the corresponding wavelength for three times. The results are shown in Figure 13 : The anti-oxidase activity of SlSPRH2 gene knockout tomato plants decreases less after high temperature treatment compared with the wild type.

[0092] (5) After analyzing the heat tolerance of SlSPRH2 gene knockout tomato plants, it is found that the oxidative damage of wild type tomato is more serious than that of SlSPRH2 gene knockout plants under high temperature stress. The results are consistent with the results in Example 3, further showing that SlSPRH2 is negatively regulated in the heat tolerance of tomato. It is shown that SlSPRH2 gene plays a significant role in improving the heat tolerance of tomato, and can provide new gene resources for breeding new varieties of heat-resistant tomato crops.

[0093] In the present application, M14 wild type of tomato Ailsa Craig (AC) background is used as material, and overexpression transgenic tomato materials of SlSPRH2, SlSPRH2 S42A and SlSPRH2 S42D are constructed, and mutant plants of SlSPRH2 gene are obtained by CRISPR / Cas9 gene editing technology. Through related heat tolerance analysis (plant phenotype analysis under high temperature stress, in vitro leaf phenotype analysis, DAB and NBT staining results of tomato leaves and anti-oxidase activity change result analysis), the present application shows the potential value of SlSPRH2 gene in improving the heat tolerance of tomato, and preliminarily reveals the core function and action path of SlSPRH2 gene and its unique Ser-42 phosphorylation site in the complex mechanism of tomato in response to high temperature stress, which opens up a new gene resource approach for future breeding of new varieties of tomato that can resist high temperature stress and maintain high yield and high quality.

[0094] The above embodiments are preferred embodiments of the present application, and are not intended to limit the present application. Without departing from the spirit or scope of the present application, technical improvements and equivalent substitutions made by those skilled in the art to the present application are within the protection scope of the present application.

Claims

1. A method for improving the heat resistance of tomatoes, characterized in that, This includes reducing the expression level of proteins with amino acid sequences such as those shown in SEQ ID NO:2, and methods for reducing expression include gene silencing or gene knockout.

2. A method for cultivating heat-resistant tomato varieties, characterized in that, This includes screening for plants with reduced gene expression, as shown in SEQ ID NO:1, or introducing a vector plasmid that knocks out the gene shown in SEQ ID NO:1 into tomato plants through genetic transformation.

3. The application of proteins with amino acid sequences as shown in SEQ ID NO:2 as detection markers in indicating the heat resistance of tomatoes by detecting their expression levels.

4. The application of genes with nucleic acid sequences as shown in SEQ ID NO:1 as detection markers in indicating the heat resistance of tomatoes by detecting their expression levels.

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